Homing endonucleases are rare-cutting DNA cleavage enzymes that are most often encoded by introns and inteins. Intron endonucleases and inteins attract considerable attention for their molecular mechanisms, phylogenetic diversity, role in genome evolution, and application in research, biotechnology and medicine. In this past funding period, we collaborated broadly to make strides in all four previous specific aims. First, we showed that phage T4 group I intron endonuclease 1-Tevl, a member of the GIY-YIG family, is bifunctional, doubling as a transcriptional autorepressor. Second, we engineered the archaeal l-Dmol intron endonuclease, a monomeric LAGLIDADG family enzyme, into a heterodimer, a homodimer and a monomer with altered specificity, an accomplishment with both practical and evolutionary implication. Third, we caught a first glimpse, by cryo-electron microscopy (EM), of a lactococcal group II intron-encoded retroelement, consisting of the protein which has endonuclease and reverse transcriptase (RT) activity, in complex with its intron RNA, scaffolded on a ribosome. Finally, we solved the structure of four variants of a mycobacterial intein, and gained insight into this intein's cleavage mechanism. With these results as a springboard, we propose the following four specific aims for the next phase of research: 1. To probe the structure and function of the l-TevI linker, which serves as a communication bridge between catalytic and DNA binding domains and controls cleavage versus repression;and to test the hypothesis that the linker acts as a redox switch, that regulates cleavage fidelity and thereby influences intron spread. 2. To use cryo-EM, among other techniques, to lay the structural foundation for biochemical function of the group II intron ribonucleoprotein (RNP), and study RNP interactions with target DNA in the tri-macromolecular initiation complex for retromobility. 3. To explore the Drosophila Penelope retro-element, which combines a GIY-YIG endonuclease with an RT related to telomerase, to determine their interrelationship, and particularly how the endonuclease communicates with a telomerase-like RT to initiate cDNA synthesis. 4. To study protein self-splicing and dispersal of a mycobacterial intein, and improve the effectiveness of intein inhibitors, which serve both as probes of mechanism and as potential anti-microbial agents. Once again we are taking a collaborative, interdisciplinary approach, combining genetics and biochemistry with computational and structural biology. In this way, we will enhance our understanding of the biology and evolution of endonucleases and inteins, as a means to promote their effectiveness as biotechnological reagents and to exploit the potential of inteins as targets for drug development.